Novel strains of bacillus thuringiensis and pesticidal composition comprising them

ABSTRACT

The present invention relates particularly to a strain of  Bacillus thuringiensis  characterized in that it expresses the gene sigma E (σE) and does not sporulate at all or little sporulates or does not produce any viable spores. The invention also relates to a pesticide composition containing said strain of  Bacillus thuringiensis.

[0001] The present invention relates to novel strains of Bacillusthuringiensis, pesticidal compositions employing them as well as the useof these strains for the expression of proteins of interest.

[0002]Bacillus thuringiensis (Bt) is a Gram-positive bacterium whichproduces proteins having insecticidal properties, especially againstlarvae of a large number of insects. These bacteria, possibly afterinactivation, are used in pesticidal compositions intended to combatinsects harmful to crops or vectors of disease, especially mosquitoes.

[0003] At present, Bt serotype 3a 3b especially is used against croppests, and the serotype H14 is used to destroy mosquito larvae.

[0004] The proteins with pesticidal activity produced by Bacillusthuringiensis are called δ-endotoxins and are produced abundantly duringsporulation. They accumulate in the form of parasporal crystallineinclusions, and can represent up to 25% of the dry weight of thesporulated cells.

[0005] Numerous genes of δ-endotoxins have been cloned, sequenced andclassified in five groups and in various subgroups on the basis ofsequence homologies and of the toxicity spectrum. The correspondinggenes are called cry genes.

[0006] Formulations based on Bacillus thuringiensis have been used asbiopesticides for close to 30 years under different trade names. The useof Bacillus thuringiensis as a biological control agent has numerousadvantages with respect to chemical pesticides; in fact, it has a narrowand very specific host spectrum and it is without effect on the insectswhich are not targets and it is without unfavorable effect onvertebrates or on the environment.

[0007] However, the slight persistence of the δ-endotoxins in theenvironment and the presence of spores in the formulations represent twodisadvantages for the marketing of products based on Bacillusthuringiensis. In order to resolve these two problems, it has beenproposed in the patent application EP-192 319 to encapsulate the toxinsin cell membranes, in particular using Pseudomonas fluorescens-typecells expressing the Cry1Ac toxin, or alternatively in the patentapplication PCT WO94/25612, by expressing the Cry1IIA toxin in anaffected nonsporulating mutant in the spoOA gene. This latter strategyis possible because the mode of expression of the cryIIIA gene isdifferent from the mode of expression of other cry genes, in fact thiscryIIIA gene is expressed from a promoter whose activation isindependent of all the genes involved in the initiation of sporulationor of the factors involved in sporulation.

[0008] In Bacillus thuringiensis, the sporulation is dependent on theexpression of two sigma factors respectively called sigma [θ]35 andsigma [θ]28; account having been taken of their great homology with thesigma (θ)E and sigma (θ)K factors in Bacillus subtilis, it is thislatter terminology which will be used below, in the same way as thecorresponding genes will be called sigE and sigK.

[0009] The present invention relates to a strain of Bacillusthuringiensis which expresses θE but does not sporulate or sporulateslittle or does not produce viable spores.

[0010] The present invention is based on the demonstration of the factthat a mutant of Bacillus thuringiensis which expresses sigE and whichdoes not express sigK produces a quantity of toxins virtually identicalto the corresponding wild strain, but, on the other hand, does notsporulate or does not produce viable spores.

[0011] This is particularly the case when the strain is a sigK⁻ strain.

[0012] The construction of such strains has two advantages: 1) avoidingthe dissemination of spores into the environment during treatment withbiopesticides; 2) increasing the persistence of the toxins in theenvironment because of their encapsulation.

[0013] Tests have shown that a Bt strain not expressing the sigma K genesigK⁻) was capable of accumulating a quantity of toxins equivalent tothe source strain, while not producing spores. It was capable ofproducing the virtual totality of the toxins encoded by the cry genes orthe related genes whose expression is dependent on the production of theθE protein.

[0014] In order to obtain sigK⁻ mutants of Bt, it is particularlyadvantageous to use an interruption technique, by insertion or deletionor change of phase of the sigK gene by introducing any DNA sequence, itbeing possible, in addition, to choose this DNA sequence so as to confera selection character on the mutant; it could be, for example,resistance to an antibiotic, especially to kanamycin, which would allowstrains which have been subjected to interruption to be selected.

[0015] The sigK⁻ mutants can likewise be obtained by the deletion of allor part of the nucleotide sequence corresponding to that of the sigKgene with or without regulatory regions.

[0016] The techniques of interruption of genes are known, they consistessentially in introducing, at the level of a DNA sequence carrying thesigK gene, any DNA sequence, the whole being introduced into the strainproduces a homologous recombination, the sigK gene being replaced by theinterrupted sigK gene. The selection character allows mutants ofinterest to be selected at this time.

[0017] Of course, it is particularly advantageous to choose, as strainsintended to be transformed, strains of Bacillus thuringiensis having avery varied or very significant toxin production. In fact, as has beenindicated above, the fact of interrupting the sigK gene only blockssporulation but does not block the production of the toxins.

[0018] Bt is understood as meaning any strain of Bacillus thuringiensis.

[0019] Thus, among the strains intended to be subjected to interruption,the industrial strains could be used. For example, Bt subsp. kurstakiHD-1 described by Dulmage H. T. (1970), or Bt israelensis, or a wildstrain such as Bt aizawai 7-29 (this strain is accessible to IEBC underthe No. T07029).

[0020] The present invention relates more particularly to the strainBacillus thuringiensis 407 SigK⁻ (pHT410) as well as the recombinantstrain Bacillus thuringiensis Kto SigK⁻ (pHTF3-1C/A (b)-IRS-T-Δ)deposited in the National Collection of Microorganism Cultures of theInstitut Pasteur, on Oct. 26, 1995 under the No. I-1634 and on Oct. 22,1996 under the No. I-1776 respectively.

[0021] It is likewise possible, in order to increase the production oftoxins, to introduce into the sigK⁻ strains according to the inventionself-replicating plasmid systems ensuring the expression of the saidtoxins according to constructs which are likewise known to the expert.INFORMATION RELATIVE TO A DEPOSITED MICROORGANISM (rule 13a of the PCT)A. The information refers to the microorganism found in the descriptionpage 3, line 32 B. IDENTIFICATION OF THE Other deposits are the DEPOSITobject of a supplementary sheet X Name of the depositing institutionINSTITUT PASTEUR Address of the depositing institution (including theZip code and the country) 28 Rue du Docteur Roux 75015 PARIS FRANCE Dateof deposition Order No. Oct. 26, 1995 I-1634 C. SUPPLEMENTARY INFORMA- Asupplementary sheet is TION (if necessary) attached for the continu-ation of this information D. INTENDED STATES FOR WHICH THE INFORMATIONIS GIVEN (if the information is not given for all the desig- natedStates) E. INFORMATION SUPPLIED SEPARATELY (if necessary) The numberedinformation below will be subsequently supplied to the InternationalOffice (specify the general nature of the information, e.g. “order no.of the deposit”) Reserved for the Reserved for the receiving officeInternational Office X This sheet has been This sheet arrived atreceived at the same the International time as the inter- Office on:national application Authorized official Authorized official J. LEPICAUD A. The information refers to the microorganism found in thedescription page 3, line 33 B. IDENTIFICATION OF THE Other deposits arethe DEPOSIT object of a supplementary sheet Name of the depositinginstitution INSTITUT PASTEUR Address of the depositing institution(including the Zip code and the country) 28 Rue du Docteur Roux 75015PARIS FRANCE Date of deposition Order No. Oct. 22, 1996 I-1776 C.SUPPLEMENTARY INFORMA- A supplementary sheet is TION (if necessary)attached for the continu- ation of this information D. INTENDED STATESFOR WHICH THE INFORMATION IS GIVEN (if the information is not given forall the desig- nated States) E. INFORMATION SUPPLIED SEPARATELY (ifnecessary) The numbered information below will be subsequently suppliedto the International Office (specify the general nature of theinformation, e.g. “order no. of the deposit”) Reserved for the Reservedfor the receiving office International Office X This sheet has been Thissheet arrived at received at the same the International time as theinter- Office on: national application Authorized official Authorizedofficial J. LE PICAUD

[0022] Form PCT/RO/134 (July 1992)

[0023] The proteins expressed by the strain could depend on the type ofpesticidal activity sought, thus Cryl is toxic for Lepidoptera, Cry1Iagainst Lepidoptera and Diptera and Cry1V against Diptera.

[0024] Generally speaking, it is possible to produce sigK⁻ of the wildstrains which naturally express a certain number of toxins such ascryIC, cryIA, cryIVA,B,D. It is likewise possible to use mutant strainssuch as described according to the invention which are sigK⁻ and whichexpress, after chromosomal or plasmid integration, genes coding forhomologous or heterologous proteins with respect to the Bt genome.

[0025] An illustration of the technique utilizable is described inBiotechnology, 1992, vol. 10, p. 418 (Lereclus et al.).

[0026] It is possible to introduce a gene, for example the cryIC gene,into the Bt sigK⁻ strain by homologous recombination. The recombinantacquires the cryIC gene and does not retain any foreign DNA.

[0027] Several toxin genes can thus be added, either in the bacterialchromosome by homologous recombination, or integrated on residentplasmids.

[0028] By way of example, a strain of Bt kurstaki at the same timeexpressing the cryIAc gene under the control of its own promoter and thecryIC gene under the control of the promoter of the cryIIIA gene.

[0029] Among the cry genes utilizable in these constructs, it ispossible to cite: cryI, cry II, cry IV and cyt.

[0030] Another method of introduction of a gene to be expressed consistsin using Gram-positive bacterial plasmids having a functionalreplication origin in Bt which is described, for example, in the patentapplication PCT WO93/02199 concerning the pHT304 and pHT315 plasmids.

[0031] The sigK⁻ strains obtained according to the process according tothe invention are utilizable, optionally after inactivation, inpesticidal compositions, in particular in insecticidal compositionsintended to be used in order to destroy larvae, in particular insectlarvae. The pesticidal compositions will be prepared according totechniques known per se, that is to say, if this is necessary, as amixture with an inert or noninert support ensuring an optimum activityof the Bacillus toxins concerned.

[0032] The inactivation of the strains, which is essential for theutilization of the sporulating strains in certain countries, is optionalin the case of the sigK⁻ mutants constructed in the context of thepresent invention. This inactivation can be carried out by any physicalor chemical method, especially by irradiation, which ensures thenon-viability of the strains. The strains according to the invention donot have any viable spores, their inactivation is easier than in thesporulated strains case.

[0033] As has been indicated above, the toxins being kept in theinterior of the bacteria allows the length of life of the toxins in theenvironment (the toxin only being liberated during the digestion of thebacterium by the larva) to be increased. However, it has been possibleto demonstrate the fact that certain mutants according to the inventionhave a resistance which is quite exceptional, in this case it isnecessary to provide for either the use of selected specific strains fortheir good digestibility in the insects to be treated, or alternativelyto provide for chemical treatments, surface-active agents for example,physical treatments, treatments with ultrasound, or biologicaltreatments, introduction of particular elements into the walls of themicroorganism (by genetic recombination technique or others) in order toensure a better digestibility or an easier accessibility of the toxinwhen the microorganism has been ingested.

[0034] This novel strain of Bt is capable of supplying one of theelements to a pesticidal composition, but is likewise useful as a vectorfor expressing homologous or heterologous genes with respect to thegenome of Bt, the said genes being cloned in the sigK⁻ mutant of Bt,either with the aid of a self-replicating plasmid, or by homologousrecombination with respect to the genome of the bacterium.

[0035] The construction of the vector system which can express, forexample, proteases, lipases or any other type of protein, may be similarto that described in the patent application PCT WO94/25612.

[0036] The invention likewise relates to a nucleotide sequencecontaining the SigE gene, not containing any active SigK gene andcontaining a sequence coding for a gene of interest.

[0037] Other characteristics and advantages of the present inventionwill appear on reading the examples below and in referring to thefigures in which:

[0038]FIG. 1 represents the interruption of the sigE and sigKchromosomal genes of Bacillus thuringiensis; the pAB1 and pAB2 plasmidsare integrated into the Bt chromosome by homologous recombination, thesecond event of homologous recombination leads to the loss of all thepRN5101 sequence; the arrows indicate the transcription direction of theAp^(R), EM^(R) and Km^(R) genes which correspond to the genesconferring, respectively, resistance to ampicillin, erythromycin andkanamycin, the triangles represent, respectively, the replication originof pBR322 (oriEc) and the replication origin of pE194ts (orits);

[0039]FIG. 2 represents the construction of the plasmids for theanalysis of transcription in Bacillus thuringiensis; pHT304-18Z is[sic], as has been previously described (Agaisse and Lereclus 1994b);the arrows indicate the transcription direction of ermC, bla and lacZand the functional replication direction in E. coli (oriEc); ori1030 isthe replication origin of the Bt pHT1030 plasmid (Lereclus and Arantes1992); the broken arrows indicate the transcription direction initiatedstarting from the promoter psigE, psigK, Bt I and Bt II, as has beenindicated above (Rong et al. 1986; Sandman et al. 1988; Wong et al.1983); the HindIII-BamHI fragments carrying the promoter regions of thespoIID, cotA and cryIAa genes have been cloned in pHT304-18Z;

[0040]FIG. 3 represents the expression of β-galactosidase in Bt underthe control of the promoters of the spoIID and cotA genes; the cells aregrown on SP medium at 30° C.; the time zero indicates the end of theexponential phase, t_(n) is the number of hours before or after timezero; A is the β-galactosidase activity of the Bt strain carryingpHTspoIID; B is the β-galactosidase activity of the Bt strain carryingpHTcotA; the specific activity of β-galactosidase is determined at thetimes indicated in Spo⁺ 407 (▪), 407 SigE⁻ () and 407 sigK⁻ (◯);

[0041]FIG. 4 represents the expression of β-galactosidase directed underthe control of cryIAa in the strains of Bt carrying pHTcryIA2, grown onSP medium at 30° C. and the β-galactosidase activity being determined atthe times indicated in Spo⁺ 407 (▪), 407 SigE⁻ () and 407 SigK⁻ (◯);

[0042]FIG. 5 represents the pHTF3-1C/A(b)-IRS-T plasmid; this plasmidderives from pBluescript II KS⁻ (the DNA of pBluescript II KS⁻ beingrepresented by the “bla+oriEc” box), it carries two sequences containingthe internal resolution site (IRS of the Tn4430 transposon (Lereclus etal., 1986) located directly on both sides of the pBluescript II KS⁻ andof a tet gene conferring resistance to tetracycline coming from Bacilluscereus, it contains, in addition, the coding part of the cryIC/A (b)chimeric gene under the control of the p3 promoter of cryIIIA (Agaisseand Lereclus, 1994) and the replication origin of the pHT1030 plasmid ofB. thuringiensis (Lereclus and Arantes, 1992);

[0043]FIG. 6 represents the recombination reaction between the two IRSsites, catalyzed by the TnpI integrase of the Tn4430 transposon presentin the Kto SigK⁻ strain, the plasmid originating from the site-specificrecombination is designated pHTF3-IC/A(b)-IRS-T-Δ.

EXAMPLE 1

[0044] Material and Methods

[0045] Bacterial Strains and Media

[0046] The strain Bt 407 (H1 serotype) and its acrystal-liferousderivative (Cry⁻ ) have been isolated by ◯. Arantes as has beendescribed above (Lereclus et al. 1989). E. coli K-12 strain TG1(Δ(lac-proAB) supE thi hsd D5 (F′traD36 pro⁺ proB⁺ laclq lacZ ΔM15)) isused for the cloning experiments (Gibson 1984). The Bt strains arecultured at 30° C. in a Luria medium (LB) and in HCT medium (Lecadet etal. 1980) or in a sporulation nutrient medium (SP medium) (Lereclus etal. 1995). The E. coli strains are cultured at 37° C. in an LB medium.The antibiotic concentration for the bacterial selection is as follows:ampicillin, 100 μg/ml (for E. coli); erythromycin, 5 μg/ml (for Bt);kanamycin, 10 μg/ml for E. coli and 200 μg/ml for Bt.

[0047] Plasmids and DNA Fragments

[0048] The pRN5101 plasmid which was supplied by S. Gruss is aheat-sensitive replication origin plasmid in Gram-positive organisms, itwas constructed by insertion of pE194ts (Villafane et al. 1987) into theClaI site of pBR322. The Bluescript plasmid (pBS KS⁻) comes fromStratagene and the pHT304-18Z and pHT410 plasmid constructs have alreadybeen described (Agaisse and Lereclus 1994b; Lereclus et al. 1989). Theoligonucleotides (Cry1A-1 and Cry1A-2) used for the PCR amplification ofthe 362 bp fragment containing the promoter region of the Cry1Aa gene(Wong et al. 1983) are described in Table 1. The Cry1A-1 primer has a 7bp extension at the 5′ end containing the HindIII restriction site andthe Cry1A-2 primer contains an 8 bp extension with the BamHI restrictionsite. The two restriction sites are introduced to facilitate cloning inpHT304-18Z. To interrupt the Bt sigE and sigK genes, the 5′ and 3′regions of the corresponding genes are amplified by PCR usingoligonucleotides with appropriate restriction sites at the 5′ end(Table 1) and sub-cloned separately in pBS KS⁻. The 5′ regions of thesigE and sigK genes are 857 and 611 bp restriction fragments ofBamHI-XbaI respectively. The 3′ regions are 807 and 606 bp EcoRI-BamHIrestriction fragments respectively. The DNA fragments containing the 5′and 3′ regions of each of the genes are purified and bound to a 1.5 kbXbaI-EcoRI fragment carrying the aphA3 gene of Enterococcus faecalis(Km^(R) cassette) (Trieu-Cuot and Courvalin 1983) in the BamHIrestriction site of the pRN5101 plasmid. The resultant heat-sensitiveplasmids pAB1 and pAB2 carry a copy interrupted by a kanamycinresistance gene in the sigE and sigK genes respectively.

[0049] The plasmids pDG675 and pDG676 respectively carrying the promoterregion of the spoIID and cotA genes of B. subtilis were supplied by Dr.P. Stragier (Institut de Biologie Physico-Chimique, Paris, France).pHTspoIID was constructed by sub-cloning the 300 bp HindIII-BamHIrestriction fragment of pDG675 between the HindIII and BamHI restrictionsites of pHT304-18Z. pHTcotA was constructed as follows: the 400 bp ofthe EcoRI-BamHI fragment of pDG676 are initially sub-cloned in pBS KS⁻,giving pKScotA. The HindIII-BamHI restriction fragment of pKScotA isthen sub-cloned between the HindIII and BamHI restriction sites ofpHT304-18Z, the resultant plasmid being designated by the name pHTcotA.

[0050] Construction and Transformation

[0051] The plasmid DNA is extracted from E. coli by the standardalkaline lysis process. The chromosomal DNA is extracted from Bt as hasbeen described previously (Msadek et al. 1990). The restriction enzymesand the T4 ligase come from New England Biolabs, Beverly, Mass. The DNAfragments are purified on agarose gel using the Prep-A-Gene kit (BioRadLaboratories, Richmond, Calif.). The oligonucleotide primers aresynthesized by Genset (Paris, France) and the PCR amplification iscarried out using the GeneAmp PCR 2400 system (Perkin-Elmer, FosterCity, Calif.). The DNA matrix used in the PCR amplficiation is eitherthe cryIAa gene already cloned from the Bt 407 strain (Lereclus et al.1989) or the chromosomal DNA extracted from the 407 Cry⁻ strain. Thereaction conditions are as follows: a 5 min. incubation at 95° C.,followed by 30 one min. cycles at 57° C. for the hybridization, one min.at 72° C. for the extension and one min. at 92° C. for the denaturation;finally, a new incubation at 72° C. for 10 min. is carried out. The Taqpolymerase comes from USB Laboratories (Cleveland, Ohio). The standardprocedure is used for the transformation of E. coli and the Bt strainsare transformed by electroporation, as has already been described(Lereclus et al. 1989).

[0052] The protein analysis is carried out after culture of the Btstrains and sonication, the analysis being carried out on 0.1% SDS −12%PAGE.

[0053] Bioassays of Insecticidal Activity

[0054] The toxicity of the preparations is estimated using the larva ofPlutella xylostella in the second stage and the free ingestion techniqueas has been described previously (Sanchis et al. 1988).

EXAMPLE 2

[0055] Construction of the SigE⁻ and sigK⁻ mutants of Bt

[0056] The pAB1 and pAB2 heat-sensitive plasmids containing the copy ofthe gene interrupted by Km^(R) of sigE and sigK respectively areintroduced into the Bt 407 Cry⁻ strain by electroporation. Thereplacement of the sigE and sigK genes by the sigE::Km and sigK::Kminterrupted copy is obtained by successive cultures of transformants inthe presence of kanamycin at a non-permissive temperature (40° C.) (seeFIG. 1). As emerges from FIG. 1, the Bt strains transformed by the pAB1or pAB2 plasmids are all resistant both to erythromycin and to kanamycinat 37° C.

[0057] The transformants in which the sigE or sigK gene has beenexchanged for its interrupted copy are cultured at a non-permissivetemperature, that is to say at which the replication of the plasmids hasbeen blocked. They can be selected by their resistance to kanamycin. TheSpo⁻ mutants (407-SigE⁻ and 407-SigK⁻ below) are resistant to kanamycinbut sensitive to erythromycin. The replacement of the Bt sigE and sigKgenes by their interrupted copy is checked by PCR analysis, thechromosomal DNA of the selected mutants is used as a matrix for the PCRand the complementary external sequences of each gene are used as aprimer in combination with sigE-4 and sigK-4 oligonucleotidesrespectively. The size of the PCR products corresponds to genesinterrupted by Km^(R).

[0058] The Bt SigE⁻ and SigK⁻ mutant strains are incapable ofsporulating. No heat-resistant spore is produced after 72 hours ofgrowth at 30° C. in HCT or SP medium. Using similar growth conditions,at least 90% of the cells of the wild strain sporulate after 24 or 48hours. The examination of the cells by phase-contrast microscopyindicates that the sigE⁻ mutant strain is blocked at an earlysporulation stage (stage II), after the formation of the asymmetricseptum dividing the mother cell and the spore compartment. The sigK⁻mutant strain is blocked in a later sporulation stage (stage IV). A grayprespore situated at one of the poles of the cell can be observed in theinterior of the cells.

[0059] The pHTspoIID and pHTcotA plasmids (see FIG. 2) carrying thepromoter regions of the spoIID and cotA genes of Bacillus subtilis fusedwith the lacZ gene are constructed to follow the appearance and thedisappearance of the θE and θK factors during the sporulation of Bt.spoIID is transcribed by an RNA polymerase containing the θE factor(Lopez-Diaz et al. 1986; Rong et al. 1986). This gene is involved in themorphological development of the spores at stage II (Young andMandelstam 1979). The cotA gene codes for a spore envelope protein(Donavan et al. 1987) and its transcription depends on θK (Sandman etal. 1988). The pHTspoIID and pHTcotA plasmids are introduced into Bt 407Cry⁻ Spo⁺, 407-SigE⁻ and 407-SigK⁻ by electroporation and the synthesisof β-galactosidase is followed during growth in SP medium (FIGS. 3A and3B). In the Spo⁺ strain, the synthesis of β-galactosidase under thecontrol of the spoIID promoter starts at t2 to attain a maximum ofapproximately 10,000 U/mg of proteins at t5 and then decreases. In thestrain in which the synthesis of β-galactosidase is under the control ofthe cotA promoter, this is detected only at t6 and attains a maximum of4000 U/mg of proteins at t11. There is no detectable expression ofβ-galactosidase (less than 10 U/mg of proteins) in the 407-SigE⁻ mutantfor the spoIID′ or cotA′-′lacZ transcriptional fusions. As thetranscription of sigK depends on θE (Sandman et al. 1988) there is noproduction of the factor K in this mutant strain. There is no detectableexpression of lacZ starting from the cotA promoter in the 407-SigK⁻mutant and the expression starting from the spoIID promoter has amaximum at t6 as in the wild strain.

EXAMPLE 3

[0060] Expression of cry1Aa′-′lacZ in SigE⁻ and SigK⁻ Mutants of Bt

[0061] To determine the temporal regulation of the promoters of thecry1Aa gene in the wild strain of Bt and in the Spo⁻ mutants, a plasmidcontaining the cry1Aa′-lacZ [sic] transcriptional fusion wasconstructed. A region containing the promoter region of the cry1Aa geneis amplified by PCR, as has been described, then cloned in pHT304-18Zupstream of the lacZ reporter gene. The resultant plasmid, designated bythe name pHTcry1A2 (FIG. 2), is introduced into the Bt 407 Cry⁻ Spo⁺,407 SigE⁻ and 407 SigK⁻ strains by electroporation. The production ofβ-galactosidase in the Spo⁺ 407 Cry⁻ strain starts at t2 and has twopeaks, the first at t7 and the second at t11 (FIG. 4). As has beenindicated for the spoIID′-‘lacZ and cotA’-′lacZ fusions, t7 and t11correspond to the maximal periods of expression of θE and θK. Theexpression of the synthesis of β-galactosidase directed by the promoterregion of cry1Aa is severely reduced in the 407-SigE⁻ mutants (FIG. 4).However, a slight β-galactosidase activity is detected at t2 with amaximum of 200 U/mg of proteins at t10. The γ-galactosidase synthesisdirected by the promoter region of cry1Aa starts at t2 and shows amaximum of 9000 U/mg of proteins at t7 in the mutant 407-SigK⁻ (FIG. 4).The second expression peak at a later sporulation time in the Spo⁺strain is not apparent in the SigK⁻ mutant, which indicates aparticipation of the θK factor in the transcription of the cry1Aa geneduring the late sporulation phase.

EXAMPLE 4

[0062] Production of the Cry1Aa Toxin in the SigE and SigK Mutants of Bt

[0063] The pHT410 plasmid carrying the cry1Aa gene of the wild strain ofBt 407 (Lereclus et al. 1989) is introduced into the Bt 407 Cry⁻ Spo⁺407-SigE⁻ and 407-SigK⁻ strains by electroporation.

[0064] The transformants are cultured on HCT and SP medium at 30° C. andthe production of crystalline inclusions is examined by phase-contrastmicroscopy and electron microscopy. After 48 hours' growth in the HCTmedium, the wide bipyramidal crystals are observed in the 407-Spo⁺ and407-SigK⁻ transformants. However, the crystals in the Spo⁺ strain areliberated while those of the SigK⁻ mutants remain encapsulated in thecell wall. Even after 72 hours' growth in the HCT medium, there is noliberation of the crystalline inclusions from the SigK⁻ mutant. Nocrystal is observed in the 407-SigE⁻ strain carrying the pHT410 plasmid.

[0065] An SDS-PAGE analysis of the proteins contained in thecrystal-cell and spore-crystal preparations from cells grown on HCTmedium show that the 407-SigE⁻ strain carrying pHT410 does not producethe Cry1Aa polypeptide of 130 kDa, unlike the 407-SigK⁻ strain carryingpHT410 which produces a toxin similar to that obtained from the 407 Cry⁻Spo⁺ strain containing the same plasmid.

[0066] The insecticidal activity of the spore-crystal and cell-crystalpreparations is analyzed using larvae of Plutella xylostella Lepidopteraat the second stage (Table 2). Having taken account of the presence ofproteins other than Cry1Aa in the 407-SigK⁻ mutant, it is not possibleto determine the precise concentration of toxin in the crystallinepreparation of this strain. This is why the LD50 is defined in terms ofculture volume to estimate the insecticidal activity of these products.The bioassays indicate that the Cry1Aa toxin produced in 407-SigK⁻ isvery toxic for the larvae of P. xylostella. However, the insecticidalactivity of these products is significantly increased by sonication.

[0067] The Bacillus thuringiensis strain, which does not express, oronly very weakly expresses, the sigma K protein under the experimentalconditions above and which is deposited at the CNCM under the No. I-1634is constructed under the following conditions:

[0068] Bacterial strain including the sigK gene is interrupted by theaphA3 gene conferring resistance to kanamycin. The strain thusconstructed in transformed by the pHT410 plasmid carrying the Cry1Aagene and the ermC gene, conferring resistance to erythromycin. Thisnonsporulating strain produces significant quantities of Cry1Aa toxinduring stationary phase la.

EXAMPLE 5

[0069] Construction of a Recombinant Strain of B. thuringiensisDesignated Kto SiqK ⁻ (pHTF3-IC/A(b)-IRS-T-Δ) Expressing a Gene Codingfor a CrY1C/Cry1A(b) Chimeric δ-endotoxin Under the Control of thePromoter of the cryIIIA Gene

[0070] The Kto strain is a natural sporulating strain of B.thuringiensis; this strain synthesizes a δ-endotoxin of Cry1A(c) type.This δ-endotoxin has an insecticidal activity against the larvae ofOstrinia nubilalis (European corn borer), a major pest of maize crops inthe United States and in Europe. This δ-endotoxin (and thus the strainKto) is, on the other hand, not very active against other importantpests belonging to the Noctuidae family such as Spodoptera littoralis,Spodoptera exigua or Mamestra brassicae (see Table 3). Conversely, theCry1C δ-endotoxin or the Cry1C/Cry1A(b) chimeric δ-endotoxin, designatedCry1C/A(b) below, whose construction (PHT81 plasmid) is additionallydescribed by Sanchis et al. (1989), are active against S. littoralis butnot very active against O. nubilalis (Table 3).

[0071] In order to increase the spectrum of activity of the Kto strain,it was of interest to introduce the cry1C gene or the chimericcry1C/A(b) gene into the Kto strain. However, it has been shown that theintroduction of a cry1-type gene (dependent on sporulation) into astrain of B. thuringiensis already containing one or more otherδ-endotoxin genes, whose expression likewise depends on sigma E andsigma K sporulation factors, is not interpreted by an increase in thetotal production of δ-endotoxins. Consequently, a recombinant straincontaining different genes of cry1 type will have a wider spectrum ofactivity but will produce less of each of the δ-endotoxins; it will thushave a lower effectiveness with respect to each of the target insectsthan strains producing a sole δ-endotoxin specific for each of thetargeted insects. This phenomenon can be explained by a titration effectof the sigma factors of sporulation by the promoters of different cry1genes present in the strain. In order to resolve this problem, it hasrecently been shown (Sanchis et al., 1996) that it is possible to placethe gene coding for the Cry1C protein under the control of the promoterof the cryIIIA gene, whose expression is independent of the sigmasporulation factors (Agaisse and Lereclus, 1994).

[0072] The cry1c gene, under the control of the cryIIIA promoter, wasintroduced into the Kto strain to give the recombinantKto(pHTF3-1C-IRS-Δ) strain (Sanchis et al., 1996). This recombinantstrain at the same time produces the Cry1A(c) and Cry1C toxins and thequantity of δ-endotoxins produced is increased by a factor of 1.5 to 2with respect to the parent strain. The increase in the total productionof the two δ-endotoxins Cry1A(c) and Cry1C obtained in the strainKto(pHTF3-1C-IRS-Δ) probably results from the fact that the expressionof the cry1C gene in this strain does not depend on specific sigmafactors of sporulation; it thus does not interfere with that of thecry1A(c) gene which is dependent on sporulation. In order to constructthe Kto SigK⁻ (pHTF3-1C/A(b)IRS-T-Δ) strain described here, the genecoding for the chimeric δ-endotoxin Cry1C/A(b) whose activity withrespect to S. littoralis is slightly superior to that of Cry1C has beenplaced under the control of the promoter of the cryIIIA gene, asdescribed previously for the cry1C gene (Sanchis et al., 1996).

[0073] Likewise, a sigK⁻ mutant (whose sigK gene is interrupted by theaphA3 gene) of the Kto strain was constructed as described in Example 2with the aid of the pAB2 plasmid (see FIG. 1). When the Kto SigK⁻strain, which is an Spo⁻ mutant of Bt, is cultured at 30° C. in HCTmedium for 48 hours, it produces significant quantities of the Cry1A(c)δ-endotoxin which accumulates in the form of a crystalline inclusionwhich remains encapsulated in the cell, which does not lyze. Theactivity of the Kto SigK⁻ strain with respect to O. nubilalis isequivalent to that of the Kto parent strain, whether the Kto SigK⁻strain has previously been sonicated or not.

[0074] The Kto SigK⁻ strain was then transformed with thepHTF3-1C/A(b)-IRS-T plasmid (see FIG. 5). This plasmid, derived frompBluescript II KS⁻, carries two sequences containing the internalresolution site (IRS) of the Tn4430_(—) transposon (Lereclus et al.,1986). These two IRSs are located directly on both sides of thepBluescript II KS and of a tet gene conferring resistance totetracycline and coming from Bacillus cereus. In addition, thepHTF3-1C/A(b)-IRS-T contains the coding part of the chimeric cry1C/A(b)gene under the control of the p3 promoter of cryIIIA and the replicationorigin of the pHT1030 plasmid of B. thuringiensis (Lereclus and Arantes,1992).

[0075] After transformation, the TnpI integrase of the Tn4430 transposonpresent in the Kto SigK⁻ strain catalyzes a recombination reactionbetween the two IRS sites and the DNA contained between these two sitesis excised. Of the two cyclic molecules resulting from thereconbination, only that which carries the replication origin of thepHT1030 plasmid and the chimeric cry1C/A(b) gene can be replicated andthe plasmid thus obtained, designated pHTF3-1C/A(b)-IRS-T-Δ, has lostthe DNA corresponding to the pBluescript II KS⁻ and to the tet gene (seeFIG. 6). The Kto SigK⁻ (pHTF3-1C/A(b)-IRS-T-Δ) recombinant strainproduces both the Cry1A(c) and the cry1C/A(b) δ-endotoxins insignificant quantity and thus has the advantage of having a widerspectrum of activity than the parent Kto or Kto SigK⁻ strain (Table 4).

[0076] In addition, such a strain has two other advantages:

[0077] 1.) The Cry1A(c) and Cry1C/A(b) δ-endotoxins remain encapsulatedin the cell. This could be interpreted by an increase in the persistenceof the toxins in the treated crop zone, because of the physicalprotection which this could confer on them against degradation and UVradiation after spreading.

[0078] 2.) The sigK⁻ mutant is an Spo⁻ mutant blocked at stage IV of thesporulation process and thus does not produce a viable spore; the use ofsuch a mutant allows the dissemination of spores into the environmentduring insecticidal treatment to be avoided. TABLE 1 Oligonucleotidesequences used as PCR primers Restriction Position site at the PrimerSequence in bp^(a) 5′ end cryIA-1 5′CCCAAGCTTGCAGGTAAATGGTTCTAAC3′136-177*  HindIII cryIA-2 5′CGCGGATCCATCTCTTTTATTAAGATACC3′ 493-518* BamHI sigE-1 5′CGGGATCCCGTTGAAAGCGTAGAGGTCAGAA3′ 16-38* BamI sigE-25′GCTCTAGAGCCAACGCGATGCATATGTTGCTA3′ 834-853* XbaI sigE-35′GGAATTCCATTGTCTGACGTGTTAGGTACA3′ 961-982* EcoRI sigE-45′CGGGATCCCGATACGCAATATCTCGCAATGA3′ 1730-1751* BamHI sigK-15′CGGGATCCCGTCCAGTTATAATTTGAGCTCCAA3′ 31-53* BamHI sigK-25′GCTCTAGAGCCCCGATTGTACCAATTGAAAT3′ 603-623* XbaI sigK-35′GGAATTCCATTAAAGCGATCGAGAGCTATT3′ 627-648* EcoRI sigK-45′CGGGATCCCGGCACCTTCTAATATTACAGATAGAA3′ 1194-1217** BamHI sigE-Ch5′TTTTCTAAAAAGCGTATTGAA3′  1-22** any sigK-Ch 5′GGAGAAACCATAGTTATGAA3′ 1-20** any

[0079] TABLE 2 Insecticidal activity of Bt strains LD50^(a) μl/ml ofpowdered Strain food^(b) 407 Spo⁺ (pHT410)  14.7 (7.9-21.8) 407-SigK⁻(pHT410) 124.6 (68.6-1358.7) 407-SigK⁻ (pHT410) sonicated  35.6(15.2-71.2) for 1 min.^(c) 407-SigK⁻ (pHT410) sonicated  9.5 (6.2-12.6)for 5 min.^(d)

[0080] TABLE 3 Activity of Cry1A (c), Cry1C and Cry1C/A (b) δ-endotoxinscompared with respect to S. littoralis and O. nubilalis LC50⁽¹⁾ withrespect to larvae of the second stage in ng of protein/cm² δ-EndotoxinsS. littoralis O. nubilalis Cry1A (c) 1000 2 Cry1C 70 >250 Cry1C/A (b) 2087

[0081] TABLE 4 Insecticidal activity of strains of Bacillusthuringiensis LC50⁽¹⁾ with respect to larvae of the second stageCharacteristics of the in ng of protein/cm² Strain strain S. littoralisO. nubilalis Kto Natural Spo⁺ strain 981 1.7 producing Cry1A (c)(758-1270) (0.9-3)   Kto (pHTF3-1C- Kto Spo⁺ strain  25 <4.2   IRS-Δ)producing Cry1A (c) (13-50)  and Cry1C Kto SigK⁻ (pHTF3- Kto⁻ Spo⁻strain  11 2.6 1C/A (b)-IRS-T-Δ) producing Cry1A (c) (3-36) (0.06-110) and Cry1C/A (b) in encapsulated form

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1 12 1 28 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 1 cccaagcttg caggtaaatg gttctaac 28 2 29 DNAArtificial Sequence Description of Artificial SequenceSynthetic DNA 2cgcggatcca tctcttttat taagatacc 29 3 31 DNA Artificial SequenceDescription of Artificial SequenceSynthetic DNA 3 cgggatcccg ttgaaagcgtagaggtcaga a 31 4 32 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 4 gctctagagc caacgcgatg catatgttgc ta 32 5 30 DNAArtificial Sequence Description of Artificial SequenceSynthetic DNA 5ggaattccat tgtctgacgt gttaggtaca 30 6 31 DNA Artificial SequenceDescription of Artificial SequenceSynthetic DNA 6 cgggatcccg atacgcaatatctcgcaatg a 31 7 33 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 7 cgggatcccg tccagttata atttgagctc caa 33 8 31 DNAArtificial Sequence Description of Artificial SequenceSynthetic DNA 8gctctagagc cccgattgta ccaattgaaa t 31 9 30 DNA Artificial SequenceDescription of Artificial SequenceSynthetic DNA 9 ggaattccat taaagcgatcgagagctatt 30 10 35 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 10 cgggatcccg gcaccttcta atattacaga tagaa 35 11 21DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA11 ttttctaaaa agcgtattga a 21 12 20 DNA Artificial Sequence Descriptionof Artificial SequenceSynthetic DNA 12 ggagaaacca tagttatgaa 20

1) Bacillus thuringiensis characterized in that it expresses the sigma E(θE) gene and does not sporulate or sporulates little or does notproduce viable spores. 2) Bacillus thuringiensis according to claim 1,characterized in that the strain does not sporulate. 3) Bacillusthuringiensis according to one of claims 1 and 2, characterized in thatit is a strain not expressing the sigma K gene. 4) Bacillusthuringiensis according to claim 3, characterized in that the SigK genehas been interrupted by introduction of a DNA sequence or has been atleast partially deleted. 5) Bacillus thuringiensis according to claim 4,characterized in that the sigK gene has been interrupted by introductionof a DNA sequence conferring a positive selection character on thestrain. 6) Bacillus thuringiensis according to claim 5, characterized inthat the positive selection character is resistance to an antibiotic. 7)Bacillus thuringiensis according to one of claims 1 to 6, characterizedin that the Bt strain expresses one or more Cry genes. 8) Bacillusthuringiensis, according to claim 7, characterized in that the Crygene(s) is/are carried by a vector, for example a plasmid. 9) Bacillusthuringiensis according to claim 7, characterized in that the Cry genesare integrated into the chromosome. 10) Bacillus thuringiensis accordingto one of claims 1 to 9, characterized in that it expresses a protein ofinterest carried by a self-replicating plasmid or by a DNA sequenceintegrated into the chromosome. 11) Bacillus thuringiensis according toclaim 10, characterized in that it contains a DNA sequence in the SigKgene expressing a protein of interest. 12) Bacillus thuringiensisaccording to claim 10, characterized in that it contains in replacementof all or part of the SigK gene a DNA sequence expressing a protein ofinterest. 13) Bacillus thuringiensis 407 SigK⁻ (pHT410) deposited at theNational Collection of Microorganism Cultures of the Institut Pasteur onOct. 26, 1995 under the number I-1634. 14) Bacillus thuringiensis KtoSigK⁻ (pHTF3-1C/A(b)-IRS-T-Δ) deposited at the National Collection ofMicroorganism Cultures of the Institut Pasteur on Oct. 22, 1996 underthe number I-1776. 15) Pesticidal composition, characterized in that itcontains a Bt strain according to one of claims 1 to
 14. 16) Compositionaccording to claim 15, characterized in that the Bt strain has beeninactivated. 17) Composition according to one of claims 15 and 16,characterized in that it has been inactivated by physical means. 18)Composition according to one of claims 15 and 16, characterized in thatit has been inactivated by irradiation. 19) Composition according to oneof claims 15 and 16, characterized in that it has been inactivated bychemical means. 20) Composition according to one of claims 15 to 19,characterized in that the Bt strain has been treated to improve thedigestibility of the strain or alternatively to improve theaccessibility of the protein. 21) Composition according to claim 20,characterized in that the Bt strain has been treated by sonication. 22)Nucleotide sequence containing the SigE gene, not containing any activeSigK gene and containing a sequence coding for a gene of interest.